Vitamin K dependent proteins are used to treat certain types of hemophilia. Classic hemophilia or hemophilia A is an inherited bleeding disorder. It results from a chromosome X-linked deficiency of blood coagulation factor VIII, and affects almost exclusively males with an incidence between one and two individuals per 10,000. The X-chromosome defect is transmitted by female carriers who are not themselves hemophiliacs. The clinical manifestation of hemophilia A is an increased bleeding tendency. Before treatment with factor VIII concentrates was introduced the mean life span for a person with severe hemophilia was less than 20 years. The use of concentrates of factor VIII from plasma and later on that of recombinant forms of factor VIII has considerably improved the situation for the hemophilia patients increasing the mean life span extensively, giving most of them the possibility to live a more or less normal life. Hemophilia B being 5 times less prevalent than hemophilia A is caused by non-functional or missing factor IX and is treated with factor IX concentrates from plasma or a recombinant form of factor IX. In both hemophilia A and in hemophilia B the most serious medical problem in treating the disease is the generation of alloantibodies against the replacement factors. Up to 30% of all hemophilia A patients develop antibodies to factor VIII. Antibodies to FIX occur to a lesser extent but with more severe consequences, as they are less susceptible to immune tolerance induction therapy.
The current model of coagulation states that the physiological trigger of coagulation is the formation of a complex between tissue factor (TF) and factor ViIa (FVIIa) on the surface of TF expressing cells, which are normally located outside the vasculature and only get accessible once an injury occurs. The complex of factor VIIa/TF activates factor IX and factor X ultimately generating some thrombin. In a positive feedback loop thrombin activates factor VIII and factor IX which then also activate factor X, the so-called “intrinsic” arm of the blood coagulation cascade, thus amplifying the generation of factor Xa, which is necessary for the generation of the full thrombin burst to achieve complete hemostasis. It was shown that by administering supraphysiological concentrations of FVIIa hemostasis is achieved bypassing the need for factor VIIIa and factor IXa. The cloning of the cDNA for factor VII (U.S. Pat. No. 4,784,950) made it possible to develop a recombinant replacement of that plasma derived coagulation factor. This factor VIIa was successfully administered for the first time in 1988 to a patient with a high titer of inhibitory antibodies to FVIII. Ever since the number of indications of factor VIIa has grown steadily showing a potential for factor VIIa to become an universal hemostatic agent (Erhardtsen, 2002). Unfortunately factor VIIa has only a plasma half-life of slightly above 2 hours and must thus be readministered frequently making such therapy invasive and very expensive. There is thus an ongoing need for improved coagulation factors, especially such that are haemostatic bypassing agents. Haemostatic bypassing agents are substances, which allow coagulation to occur when administered to patients in whom certain coagulation factors are missing, non-functional or blocked by inhibitory antibodies. The activity of such compounds to bypass blocks in the coagulation cascade (haemostatic bypassing activity) can be measured by coagulation assays known in the art. Essentially haemostatic bypassing agents have the ability to activate substrates of a missing, non-functional or blocked coagulation factor or other substrates in the coagulation cascade “downstream” of the missing, non-functional or blocked coagulation factor in a direct way such that the missing, non-functional or blocked coagulation factor is no longer needed for effective thrombin generation.
Also factor X has been the subject of extensive research.
The cDNA for factor X has been characterized (Leytus et al. 1984, PNAS, 82: 3699-3702). Coagulation factor X is a vitamin-K dependent glycoprotein of a molecular weight of 58.5 kDa, which is secreted from liver cells into the plasma as a zymogen. Initially factor X is produced as a prepropeptide with a signal peptide consisting in total of 488 amino acids. The signal peptide is cleaved off by signal peptidase during export into the endoplasmatic reticulum, the propeptide sequence is cleaved off after gamma carboxylation took place at the first 11 glutamic acid residues at the N-terminus of the mature N-terminal chain. A further processing step occurs by cleavage between Arg182 and Ser183. This processing step also leads concomitantly to the deletion of the tripeptide Arg180-Lys181-Arg182. The resulting secreted factor X zymogen consists of an N-terminal light chain of 139 amino acids (Mr 16,200) and a C-terminal heavy chain of 306 amino acids (Mr 42,000) which are covalently linked via a disulfide bridge between Cys172 and Cys342. Further posttranslational processing steps include the β-hydroxylation of Asp103 as well as N- and O-type glycosylation.
Both factor VIIIa/factor IXa or factor VIIa/TF are under physiological conditions able to activate factor X on activated platelet surfaces by cleaving carboxy-terminal to Arg234, thus liberating the so called activation peptide of 52 amino acids from Ser183 to Arg234.
In an autocatalytic cleavage activated factor X (factor Xa) cleaves off a small fragment at the C-terminal end of its heavy chain carboxy-terminal to Arg464 leading to factor Xaβ. However the physiological relevance of this cleavage is not clear as both forms of factor Xa have comparable catalytic activities.
Several attempts have been made to modify factor X:
Wolf et al. 1991 (JBC. 266, no. 21. pp. 13726-13730) deleted the activation peptide of factor X replacing it with the dipeptide Arg-Lys which leads to the introduction of 2 novel furin cleavage consensus sites within the region of the activation peptide of factor X. Such factor X variants are activated during intracellular processing leading thus to the secretion of activated factor X.
Wolf et al. 1995 (Blood. 86, pp 4153-4157) produced acylated inactive variants of factor Xa, which are slowly deacylated after injection into blood plasma thereby generating activated factor X over time.
Rudolph et al. 1997 (Prot. Express and Puri., 10: 373-378), modified factor X in the region of the propeptide cleavage site and found that replacement of Thr39 by Arg improved the efficacy of propeptide processing in cell culture considerably.
Camire et al. 2000 (Biochemistry. 39 pp. 14322-14329) achieved a higher degree of gamma carboxylation in cell culture by replacing the prepropeptide of factor X by that of thrombin. However though the rate of gamma carboxylation was increased 10-30% of factor X remained uncarboxylated.
Rudolph et al., 2002 (Thromb Haemost., 88:756-62) created factor X variants with deleted activation peptide. It could be seen that such factor X variants were auto-activated in a cofactor independent way and the paper concludes that the primary function of the activation peptide is to prevent spurious activation of FX.
Thiec et al. 2003 (JBC, 12, pp 10393-10399) replaced the Gla domain and the first EGF domain of factor X with the corresponding domain of FIX to investigate the ability of such chimeras to interact productively with the TF/FVIIa complex.
WO 98/38317 (Priority: 27 Feb. 1997) claims factor X analogues with a modification at the site of the natural activation cleavage site between Gly228 and IIe235 such that proteases which do not naturally activate factor X can cleave and activate such factor X analogues.
WO 98/38318 (Priority: 27 Feb. 1997) teaches factor X analogues in which amino acids Arg180 to Arg234 are deleted and amino acids from Gly173 to Arg179 are modified such that proteases, which do not naturally activate FX, can cleave the modified sequence thus activating the factor X analogues described above.
WO 01/10896 (Priority: 10 Aug. 1999) describes factor X analogues, which have substitutions of at least one of the amino acids between Glu226 and Me235. In the example the introduction of a FIX derived activation cleavage site is shown which makes the factor X variant cleavable by FXI.
WO 03/035861 (Priority: 19 Oct. 2001) claims variants of factor X in which the activation peptide has been removed and replaced by the amino acids P10 to P1 of fibrinopeptide A creating a chimeric thrombin cleavage site rendering this factor X variant activatable by thrombin.
WO 2004/005347 (Priority: 3 Jul. 2002) teaches variants of factor X which can be activated by thrombin by modifying the residues P3-P2-Pi-Pi′-P2′-P3′which is in wild type factor X Leu-Thr-Arg-Ile-Val-Gly (residues 232-237 of SEQ ID NO: 2) to X-Pro-Arg-Ala-Y-Z.
Volkel et al (2005), Mol. Biotechnol., 29 (1):19-30 teaches the introduction of a novel protease cleavage site in the FX activation peptide such that prostrate specific antigen specifically activates said FX variant.
Though some authors suggested that activated factor X (FXa) might be used as a haemostatic bypassing agent (Ni et al., 1992 (Thromb. Haemost. 67:264-271); Himmelspach et al., 2002 (Thromb. Haemost. 88:1003-1011)) some concerns remain that such pharmaceutical preparations might be thrombogenic and could lead to disseminated intravasal coagulation (DIC).
The therapeutic use of the non-activated zymogen factor X appears to be a much safer approach. U.S. Pat. No. 4,501,731 (priority 27 Jun. 1983) suggests the use of factor X as a haemostatic bypassing agent on its own. In WO 03/006054 (Priority: 10 Jul. 2001) it has been shown in addition that factor X in pharmaceutical compositions is able in combination with FVIIa to enhance the haemostatic efficacy of FVIIa synergistically.
However, as the efficacy of activation of factor X via the intrinsic pathway of coagulation is severely compromised in inhibitor patients whereas the extrinsic pathway of coagulation (due to the restricted availability of tissue factor) seems to be limited to the initiation phase of coagulation it is of advantage to modify factor X in such a way to facilitate its activation in situations in which coagulation is needed and bypassing the need of cofactors of limited availability and/or activity. The variant factor X zymogen must be stable so that it can be produced and administered without activation but that in case coagulatory activity (e.g. thrombin generation) is needed, activation occurs at higher rates without the need of the natural activators of the intrinsic and the extrinsic pathway of coagulation.
It has been described that several authors attempted to generate factor X variants which can be activated by proteases not naturally cleaving and activating FX. These factor X variants either consisted of deletions of the activation peptide and/or the modification of the sequence of the activation peptide preceding the cleavage site at Arg234 optionally also allowing the modification of IIe235. As it has also been shown (Rudolph et al., 2002 (Thromb Haemost., 88:756-62) that a primary effect of the activation peptide of factor X is to prevent autoactivation to FXa, factor X variants with deletions and modifications of the activation peptide are susceptible to premature activation. Pharmaceutical compositions comprising such FX variants might therefore entail a thrombogenic risk.
One problem addressed in the present invention is to identify haemostatic bypassing agents. In particular, there is a need for haemostatic bypassing agents, which can be used to treat patients having a high titer of factor VIII inhibitors.